Organization of the mouse Ruk locus and expression of isoforms in mouse tissues
Ruk is a recently identified gene with a complex pattern of expression in mammalian cells and tissues. Multiple Ruk transcripts and several protein isoforms have been detected in various types of cells. Ruk proteins have multidomain organization characteristic of adapter proteins involved in regulation of signal transduction. Interaction of some Ruk isoforms with several signalling proteins, including the p85 regulatory subunit of the Class IA PI 3-kinase, c-Cbl and Grb2, has been demonstrated. Ruk(l), an isoform with three SH3 domains, inhibits lipid kinase activity of the PI 3-kinase in vitro; overexpression of this protein induces apoptotic cell death of primary neurons in culture and changes in membrane trafficking in other cultured cells. However, shorter isoforms of Ruk block pro-apoptotic effect of Ruk(l), suggesting that expression of different combinations of Ruk proteins in cells could be involved in the regulation of their survival and other intracellular processes. To understand the mechanism of differential expression of Ruk proteins we studied organization of the mouse Ruk gene and its transcripts. Twenty-four exons of the Ruk gene span over 320 kb of the mouse chromosome X. Analysis of cDNA clones, ESTs and products of RT-PCR amplifications with different combinations of primers revealed how alternative splicing and promoter usage generate a variety of Ruk transcripts and encoded protein isoforms in different mouse tissues.
[Show abstract] [Hide abstract] ABSTRACT: Conditional deletion of the tumour suppressor gene Apc within the murine intestine results in acute Wnt signalling activation. The associated over-expression of a myriad of Wnt signalling target genes yields phenotypic alterations that encompass many of the hallmarks of neoplasia. Previous transcriptomic analysis aimed at identifying genes that potentially play an important role in this process, inferred the Hormonally upregulated Neu-associated kinase (HUNK/Mak-v/Bstk1) gene as a possible candidate. Hunk is a SNF1 (sucrose non fermenting 1)-related serine/threonine kinase with a proposed association with many different tumour types, including colorectal cancer. Here we describe the generation of a novel Hunk kinase deficient mouse which has been used to investigate the involvement of Hunk-kinase activity in intestinal homeostasis and tumourigenesis. We show that in the morphologically normal intestine, Hunk-kinase negatively regulates epithelial cell proliferation. However, the increase in cell proliferation observed in the Hunk kinase deficient intestine is counteracted by increased cell migration, thereby maintaining intestinal homeostasis. Using qRT-PCR, we further demonstrate that Hunk is significantly over-expressed in Apc deficient / Wnt-signalling activated intestinal tissue. Using the classical intestinal tumourigenesis Apc (Min) mouse model we show that loss of Hunk-kinase activity significantly reduced tumour initiation rates in the small intestine. However, an accompanying increase in the size of the tumours counteracts the impact this has on overall tumour burden or subsequently survival. In the intestinal setting we demonstrate that Hunk has a role in normal intestinal proliferation and homeostasis and, although it does not alter overall survival rates, activity of this kinase does impact on tumour initiation rates during the early stages in tumourigenesis in the small intestine.0Comments 0Citations
- "Total RNA extraction and first-strand cDNA synthesis were carried out as described previously . For analysis of Hunk expression in mouse tissues one μl of cDNA was used as a template for PCR amplification with primers 5′- agatccagcagatgatccgac-3′ and 5′-tagcgctcaagtttcttgttcaa-3′ and Platinum AccuPrime DNA polymerase (Invitrogen). "
[Show abstract] [Hide abstract] ABSTRACT: Amyotrophic lateral sclerosis (ALS) is characterised by substantial loss of both upper and lower motor neuron function, with sensory and cognitive systems less affected. Though heritable forms of the disease have been described, the vast majority of cases are sporadic with poorly defined underlying pathogenic mechanisms. Here we demonstrate that the neurological pathology induced in transgenic mice by overexpression of γ-synuclein, a protein not previously associated with ALS, recapitulates key features of the disease, namely selective damage and loss of discrete populations of upper and lower motor neurons and their axons, contrasted by limited effects upon the sensory system.0Comments 15Citations
- "Previously described protocols were used for Western blotting (Buchman et al., 2002). Protein levels were quantified using Cy3-or Cy5-conjugated secondary antibodies (Invitrogen, CA) and FluorChem Q MultiImage III system (Cell Biosciences, CA). "
[Show abstract] [Hide abstract] ABSTRACT: MAK-V/Hunk is a scantily characterized AMPK-like protein kinase. Recent findings identified MAK-V as a pro-survival and anti-apoptotic protein and revealed its role in embryonic development as well as in tumorigenesis and metastasis. However molecular mechanisms of MAK-V action and regulation of its activity remain largely unknown. We identified Nedd4 as an interaction partner for MAK-V protein kinase. However, this HECT-type E3 ubiquitin ligase is not involved in the control of MAK-V degradation by the ubiquitin-proteasome system that regulates MAK-V abundance in cells. However, Nedd4 in an ubiquitin ligase-independent manner rescued developmental defects in Xenopus embryos induced by MAK-V overexpression, suggesting physiological relevance of interaction between MAK-V and Nedd4. This identifies Nedd4 as the first known regulator of MAK-V function.0Comments 2Citations
- "Proteins were resolved in SDS-PAGE and transferred to Hybond P membrane (GE Healthcare, Little Chalfont, UK). Membranes were incubated with primary rabbit polyclonal anti- FLAG (Sigma, USA), anti-Nedd4 (Abcam, UK), anti-Nedd4-2 , or anti-neomycin phosphotransferase II (Millipore, USA), mouse monoclonal anti-a-tubulin, clone DM1A antibodies (Sigma , USA) or affinity purified rat anti-MAK-V antibodies  followed by secondary anti-rabbit, anti-mouse or anti-rat horseradish peroxidase-conjugated antibodies (HRP; GE Healthcare, USA) as described previously . To detect GST, anti-GST HPR conjugate (GE Healthcare, USA) was used. "